Sophisticated mechanisms have evolved in mammals to repair the body in the event of vascular injury and so maintain hemostasis. The injured blood vessel constricts to reduce the blood flow to the area, platelets aggregate to reduce the loss of blood from the area, and fibrinogen is cleaved to produce fibrin which then polymerises and forms a clot. This clot covers the area of vascular damage, preventing blood loss. Polymerised fibrin also provides a provisional matrix which enhances the subsequent repair process. Once the blood vessel has been repaired the clot dissolves. The process leading to the formation of the clot is the coagulation cascade, and the process leading to its dissolution is the fibrinolysis cascade. Imbalances in the blood coagulation process are thought to be at the origin of a large and disparate number of disease conditions, which are linked by an unwanted build up of fibrin. The scale of fibrin build up is determined by the delicate equilibrium between the two biochemical cascades in the human body. Agents that can modulate the balance between coagulation and fibrinolysis are therefore potentially valuable in the treatment of these disease conditions.
Studies have shown that coagulation and fibrinolysis are linked through the generation of α-thrombin. α-Thrombin is the final product of the blood coagulation cascade and is responsible for the conversion of fibrinogen into fibrin. In addition to mediating coagulation, α-thrombin also reduces the rate at which blood clots are broken down by the serine protease plasmin. The protein that mediates this antifibrinolytic effect of α-thrombin is TAFI (Thrombin Activatable Fibrinolysis Inhibitor).
TAFI is a 60 kDa glycoprotein found in human plasma. It is also known as procarboxypeptidase B, carboxypeptidase B, plasma carboxypeptidase B, carboxypeptidase U and carboxypeptidase R. Following initiation of the coagulation cascade it is transformed into an activated form, TAFIa, whereupon it acts upon the fibrin matrix of the developing blood clot to prevent its dissolution. TAFI circulates in normal plasma at a concentration of about 75 nM in an inactive form. Thrombin converts the inactive zymogen to the active TAFI (TAFIa), a reaction that is augmented about 1250-fold by thrombomodulin. Once activated, TAFIa cleaves both C-terminal arginine and lysine residues from the developing fibrin clot. The removal of these dibasic amino acids from the surface of the fibrin matrix attenuates clot lysis by inhibiting the binding of the key mediators of fibrinolysis: tissue plasminogen activator (tPA) and its substrate, plasminogen, which is the precursor of plasmin. Both tPA and plasminogen contain a structural motif called a kringle domain which binds tightly to C-terminal lysine residues. The removal of these binding sites prevents the formation of a ternary complex between tPA, plasminogen and fibrin and this inhibits the conversion of plasminogen to plasmin, thus protecting the clot from rapid degradation.
In the presence of a TAFIa inhibitor, TAFIa will not be able to act upon a developing fibrin clot as described above to inhibit fibrinolysis of the clot. Thus a TAFIa inhibitor should serve to enhance fibrinolysis.
It can be seen that, in pathologies where the normal equilibrium between coagulation and fibrinolysis is disturbed in favour of coagulation, there will be a larger amount of fibrin present than normal. This makes it more likely that the subjects will develop one or more of the conditions in which thrombus build up is implicated. Such subjects can be expected to benefit from treatment with a pro-fibrinolytic agent. McKay et al. (Biochemistry 1978, 17, 401) disclose the testing of a number of compounds as competitive inhibitors of bovine carboxypeptidase B of pancreatic origin. Inhibition was measured by the inhibitor's efficiency in protecting the active centre tyrosine and glutamic acid of bovine carboxypeptidase B from irreversible alkylation by bromoacetyl-D-arginine or bromoacetamidobutylguanidine. It is suggested that such inhibitors could act as bradykinin potentiators. Bovine enzymes of pancreatic origin are very different to those found in human plasma, so one would not expect inhibitors of one to inhibit the other. Moreover, such inhibitors are directed towards a very different utility. Accordingly this disclosure provides no teaching of TAFIa inhibitors or their utility.
Redlitz et al. (J. Clin. Invest. 1995, 96, 2534) teach the involvement of plasma carboxypeptidase B (pCPB, or TAFI) in the formation of clots. The lysis of blood clots was followed in the absence and presence of pCPB, whereupon it was found that the presence of pCPB slowed clot lysis. To confirm that pCPB was responsible two control reactions were run; one where the lysis experiment was repeated in the presence of pCPB and potato carboxypeptidase inhibitor, PCI, and a second where the lysis reaction was conducted in the presence of plasma from which pCPB was removed. In both cases lysis proceeded uninhibited.
Boffa et al. (J. Biol. Chem. 1998, 273, 2127) compare plasma and recombinant TAFI and TAFIa with respect to glycosylation, activation, thermal stability and enzymatic properties. Inhibition constants for three competitive inhibitors were determined: ε-aminocaproic acid (ε-ACA), 2-guanidinoethylmercaptosuccinic acid (GEMSA) and potato carboxypeptidase inhibitor (PCI).
There are large numbers of carboxypeptidases (i.e. enzymes that cleave the C-terminal amino acid from a peptide). They may be classified as acidic, neutral or basic, depending on the type of amino acid they cleave. Basic carboxypeptidases cleave arginine, lysine and histidine. TAFIa is a member of a specific subset of the basic carboxypeptidases. In terms of the present invention, the inhibitors disclosed above by Redlitz et al. and Boffa et al. are too weak, non-specific or otherwise unsuitable to be considered as suitable TAFIa inhibitors for therapeutic application. Further, whilst the role of TAFIa in clot lysis is explained, there is no suggestion that TAFIa inhibitors can be used to treat disease.
U.S. Pat. No. 5,993,815 teaches the use of a peptide that binds to the TAFI zymogen, thereby inhibiting its activation, to treat those disorders where a C-terminal lysine or arginine is cleaved from an intact peptide. Suitable disorders are arthritis, sepsis, thrombosis, strokes, deep vein thrombosis and myocardial infarctions. The peptide used is an antibody or a functionally active fragment. The peptide should be used in an amount to promote fibrinolysis in vivo.
WO00/66550 and WO00/66557 disclose broad classes of compounds useful as inhibitors of carboxypeptidase U. Inhibitors of carboxypeptidase U are postulated to facilitate fibrinolysis and thus the compounds are taught as useful in the treatment of thrombotic conditions. There is no data to support this assertion, though details of a suitable assay are given.
WO00/66152 discloses formulations containing a carboxypeptidase U inhibitor and a thrombin inhibitor. Suitable carboxypeptidase U inhibitors are those of WO00/66550. The formulations are taught as primarily useful in treating thrombotic conditions.
WO01/19836 discloses a series of phosphonate esters and analogues thereof as carboxypeptidase B inhibitors that are suitable for the treatment or prevention of thrombotic diseases.
WO02/14285 discloses a series of α-imidazolylmethyl-ω-aminocarboxylic acids and Nα-(ω-aminoalkyl)-histidine derivatives that are inhibitors of TAFIa. The compounds are considered to be potentially useful in the treatment of a number of conditions.
The present invention discloses a further class of TAFIa inhibitors.